Aim 1: Investigate how material composition dictates mechanical properties. Our initial studies centered on adhesives formed by polydextran aldehyde (PDA) and the model amine-containing protein, bovine serum albumin (BSA), as well as polyethylenimine (PEI), a high-content amine polymer. We systematically studied how PDA chain length, aldehyde content, and the aldehyde/amine (CHO/NH2) network ratio influenced the rate of material formation, cohesive, and adhesive properties of resultant materials. We found that each of these parameters can be varied to prepare bioadhesives that range in storage moduli from 102-105 Pa and adhesive strengths from 1-6 kPa, which is on the order of clinical adhesives. In addition, the rate of material formation can be tuned from seconds to minutes after delivery by varying the (CHO/NH2) ratio. Further, degradation can be tuned from days-to-years. Aim 2: Investigate mechanism of material formation and hydrolysis. In vitro and in vivo degradation studies of PDA-based adhesives employing eGFP and IL-2, an important protein immunoregulator, indicated that along with pure protein, proteins heavily functionalized with permanently ligated fragments of dextran were also released. If protein had been crosslinked into the PDA network via pure imine chemistry as intended, then hydrolysis-based degradation should have released only native protein. Analytical studies revealed that in addition to imine formation, a time-dependent Maillard reaction was occurring. The reaction begins with imine crosslinking, followed by an Amadori rearrangement affording non-hydrolyzing keto amines that react further affording a complex mixture of Maillard products. Aim 3: Develop adhesives towards clinical applications. Delivery of doxorubicin (DOX): We prepared a multicomponent adhesive by mixing DOX, PDA, and BSA. The adhesive is mechanically rigid (10kPa) and adheres avidly to tissue (adhesive stress 4kPa). This DOX-PDA-BSA adhesive slowly hydrolyzes (2 months), locally releasing free DOX, DOX-PDA and 11nm particles composed of all three components that enter cells, localize to the cytoplasm and kill A549 lung carcinoma cells With respect to the cell penetrating peptides: Aim 1. Determine cellular mechanisms of resistance towards SVS-1. We undertook a systematic investigation to determine if cancer cells can develop resistance. Our report is the first to show that eukaryotic cells can, indeed, develop resistance to ACPs. In collaboration with Dr. Klar (CCR), we first utilized S. pombe fission yeast to rapidly identify three different loss-of-function gene mutations that conferred resistance to SVS-1. Genetic and mechanistic studies in yeast facilitated discovery of similar mechanisms operating in mammalian cancer cells. Interestingly, both organisms developed resistance by altering their cell-surface glycans rather than directly changing the composition of their lipid membranes, the target of ACP action. Changes in glycosylation (pyruvylated galactose for yeast and sialic acid for human cancer cells) led to a reduction in the electrostatic charge at the cell surface. This, in turn, reduces the potential of peptide to accumulate at the surface of cells and confers resistance. Given the plethora of studies using model membranes to study ACPs, our work shows that the role of glycans may have been underappreciated. Aim 2: Modulating the activity of SVS-1. The mechanisms by which SVS-1 interacts with cells are complex and dependent not only on the peptide's structural features, but also on the concentration of peptide presented to cells. When cells are presented with concentrations of SVS-1 that are below its lytic IC50 ( 5 uM), the peptide engages the membrane, but is not lytic. Rather, it rapidly enters cells, accessing the cytoplasm and over time, the nucleus. Thus, SVS-1 is both a lytic ACP and a cell penetrating peptide (CPP), depending on concentration. As a CPP, SVS-1 enters cells by both direct translocation through the membrane and clatherin-dependent endocytosis. Although we exploit SVS-1 as a CPP, some delivery applications are complicated by the narrow concentration window differentiating its lytic and cell penetrating activities. Further, it would be optimal if SVS-1 entered cells purely by direct translocation, thus avoiding issues of endosomal escape. Decoupling and isolating the activity and cellular entry mechanisms of membrane-perturbing peptides by molecular design is extremely challenging. However, over the course of studying SVS-1, we discovered a correlative relationship between the peptide's folding propensity and both its lytic activity and its propensity for endocytic uptake; structural derivatives of SVS-1 that show a higher propensity to fold are more lytic as ACPs and are endocytosed more readily as CPPs. Based on this observation, we hypothesized that if an intrinsically disordered membrane-active peptide could be designed, it should avoid endocytosis, enter cells via direct translocation, and be less cytotoxic. We designed a peptide, CLIP6, which contains a key glutamate residue in its sequence that disrupts the amphiphilicity of the peptide rendering it incapable of folding. We also showed that CLIP6 is intrinsically disordered and exclusively enters cells by non-endosomal mechanisms, while being remarkably cytocompatible and serum-stable. Further, CLIP6 can deliver membrane-impermeable cargo directly to the cytoplasm of cells. Aim 3: Exploiting SVS-1 and its derivatives towards therapeutic applications. Small molecule delivery: Many lead molecules identified in drug discovery campaigns are eliminated from consideration due to poor solubility and low cell permeability. These orphaned molecules could have clinical value if solubilized and delivered properly. In an initial assessment of SVS-1's utility, the model hydrophobic drug Paclitaxel (PTX) was ligated via a self-immolative linker, increasing its solubility by 1000-fold. SVS-1 successfully delivered and released PTX to cancer cells in vitro and in vivo, where tumor burden was significantly reduced in a xenograft mouse model. Protein delivery: Biologics have made tremendous impact clinically despite the fact they can currently address only cell surface targets. The ability to deliver biologics into cells would vastly increase their utility. CPPs, such as TAT, have been used to deliver biologics, but nearly all enter cells through some degree of endocytosis, limiting their effectiveness. We developed a family of expression-capable cell-penetrating peptides (XCPs) based on SVS-1 and CLIP6, which replace their D-proline turn with sequences containing all L-residues. XCPs can be directly fused to proteins recombinantly, eliminating the need for chemical ligation. We are currently optimizing our first-generation designs, which show equal partitioning between direct translocation and endocytic uptake mechanisms.